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PubMed1983

Characterization of lipopolysaccharide from a heptoseless mutant of Escherichia coli by carbon 13 nuclear magnetic resonance.

Lipopolysaccharide (LPS) isolated from Escherichia coli D31m4, a heptoseless mutant, was studied by 13C and 31P NMR spectroscopy. Modified isolation and purification procedures are described which permitted high resolution NMR spectra to be obtained from samples of intact LPS. 31P NMR was used to monitor the purity and native heterogeneity of LPS samples. The anomeric carbon region of the 13C NMR spectrum taken at pH 7 contained five resonances that were assigned on the basis of chemical shift correlation, 13C-1H couplings, and T1 relaxation times. Two resonances, at 99.9 and 100.8 ppm, were attributed to two residues of 3-deoxy-D-manno-octulosonate (KDO) of which both were tentatively assigned to the alpha configuration. The Lipid A moiety gave rise to resonances at 94.0 and 94.9 ppm, both assigned to GlcNI, and a resonance at 103.1 ppm, assigned to GlcNII. The two anomeric carbon resonances observed for GlcNI reflected the variable substitution of C-1 with monophosphate or diphosphate groups. GlcNI and GlcNII were ascertained to be of the alpha and beta anomeric configuration, respectively, through chemical shift comparisons with model saccharides. The accepted KDO linkage site at C-3' of GlcNII appears not to be supported by the 13C chemical shift data.

Carbohydrates↗
PubMed1983

Complete structure of lipid A obtained from the lipopolysaccharides of the heptoseless mutant of Salmonella typhimurium.

A highly purified monophosphoryl lipid A, TLC-3 fraction obtained from the lipopolysaccharides of the heptoseless mutant Salmonella typhimurium G30/C21 was converted to the dimethyl pentatrimethylsilyl derivative and analyzed by proton NMR spectroscopy at 400 MHz. Substantial downfield shifts of the resonances for protons at the 3- and 3'-carbons of the glucosamine disaccharide to 5.06 and 5.15 ppm, respectively, occurred from the normal range of 3.5-4.1 ppm, indicating that these two positions on the sugar rings were acylated. Significant downfield shift of the resonances for protons at the 4- and 6'-carbons did not occur, indicating the absence of acyl groups at these two positions. Since positive ion fast atom bombardment mass spectrometry previously established the presence of hydroxymyristoyl and myristoxymyristoyl esters at the reducing end and distal subunits, respectively, these acyl groups must be attached to the oxygen of the corresponding 3- and 3'-carbons of lipid A. With these results, we can now describe the complete structure of the monophosphoryl lipid A, TLC-3 from S. typhimurium.

Heptoses↗
PubMed1983

Structure and metal-binding properties of lipopolysaccharides from heptoseless mutants of Escherichia coli studied by 13C and 31P nuclear magnetic resonance.

The structure and metal-binding properties of lipopolysaccharides (LPS) from heptoseless mutants of Escherichia coli were studied by 13C and 31P NMR techniques. Carbon-13 NMR spectra were used to determine the linkages and configurations of the saccharide backbone and the types and locations of fatty acyl groups in E. coli LPS. Resonance assignments for native LPS were made by chemical shift correlation with model compounds, deacylated LPS, lipid A, deacylated lipid A, and fatty acids released from LPS by mild alkaline hydrolysis. The 3-deoxy-D-manno-octulosonate (KDO) disaccharide was tentatively assigned the structure KDO alpha 2 leads to 5KDO alpha 2 leads to. The presence of amide- and ester-linked 3-hydroxy and 3-acyloxy fatty acids in native LPS was confirmed directly from the 13C spectrum and evidence is presented for a labile acyl ester at C-3' (GlcNII) of the lipid A moiety. A significant finding was that the KDO disaccharide is linked to the C-6' position of the lipid A moiety, rather than C-3', as previously reported. The effects of binding Ca2+, Cd2+, Yb3+, Gd3+, and La3+ on the 31P NMR spectrum of LPS indicated that the glycosidic diphosphate moiety participates in a high affinity metal-binding site.

Cadmium↗
PubMed1982

Effect of endotoxin and glucocorticoid pretreatment on hexose monophosphate shunt activity in rat liver.

The acceleration of glycolysis by the Embden-Meyerhof pathway (EMP) in endotoxic and septic states and its counteraction by glucocorticoids has been demonstrated by past research. Although the glycolytic contribution of the hexose monophosphate (HMP) shunt is minor, its response during endotoxemia, if similar to that of EMP, could be theoretical interest, Fasted male rats (150-260 gm) were sacrificed at 5 hr after IV injection of E coli endotoxin in dosages of 2 or 3 mg/100 gm rat weight: LD50 (Nm= 15). A second group received 1 mg dexamethasone (DMS) IV per 100 gm rat weight simultaneously with endotoxin (N = 15). Livers were homogenized in 0.25 M cold sucrose and centrifuged at 15,000 g for 20 min. Specific activity of glucose-6-phosphate dehydrogenase (G6PDH) in control livers (N = 17) was 7.1 nmoles of substrate consumed per min/mg biuret protein. Endotoxin raised G6PDH activity by 49% to 10.64 units, and the endotoxin-DMS-protected group was 6.0 units. Levels of 6-phosphogluconate (6PG) were also measured in frozen liver biopsies from similar groups of rats. Liver 6PG concentrations of control (N = 15), endotoxic (N = 15), and endotoxified-DMS-treated (N = 9) groups were 22.5, 14.3, and 17.6 nmoles/gm wet tissue, respectively. The data indicate a significant 36% acceleration in 6PG consumption during endotoxemia, which is not blocked by DMS. The cofactor, nicotinamide adenine dinucleotide phosphate (NADP), decreased significantly by 18% from the control level of 152 nmoles/gm liver (N = 9) during endotoxemia, and this fall was not corrected by DMS. In a small group (N = 6), sedoheptulose-7-phosphate declined from the control value of 76 nmoles/gm wet liver by 38% after endotoxification. It is concluded that endotoxin stimulates G6PDH, the initial enzyme of the HMP pathway, and accelerates consumption of several intermediates, Glucocorticoid prevents the enzyme activity increase but does not restore 6PC and NADP concentrations to normal levels, suggesting that different enzyme sites along the HMP shunt may have unequal responses to DMS.

Animals↗
PubMed1980

[Action of soluble carbohydrates from avocado (Persea gratissima Gaertner) fruit on utilization of calcium in the rat].

Ethanolic extract containing soluble sugars was prepared from avocados (variety americana) coming from Madagascar. In this extract, thin layer and paper chromatography showed the presence of a ketoheptose, the mannoheptulose, in amount of 0.1 g/ml extract or 1.1 g/100 g fresh pulp. Physiological experiments were performed with avocado extract and comparatively with a sample of commercial mannoheptulose. Six-month-old rats were given by stomach tube 10 mM CaCl2 solution (+ 45Ca) containing a carbohydrate. Depending on the group, the carbohydrate was 20 and 100 mM glucose (control), D-mannoheptulose 200, 100, 50 and 25 mM, avocado extract concentrated or diluted 1/2, 1/4, 1/8, 1/16. Blood samples were taken at different times after the administration and plasma radioactivity was measured. Rats were sacrificed at 24 hours; femur and axis radioactivity was used as a measure of calcium absorption. Both mannoheptulose and avocado extract were potent on calcium absorption. This absorption increased progressively with sugar concentration, in mannoheptulose and avocado groups. The interpretation of these results was discussed and also their application to the Nutrition in tropical and subtropical countries.

Absorption↗
PubMed1977

The effects of calcitonin on pancreatic exocrine secretion in dogs.

The effect of calcitonin on pancreatic secretion was studied in unanesthetized dogs with a Thomas cannula implanted opposite the main pancreatic duct. Pancreatic juice was collected for a 60 minute control period, during which time secretion was stimulated by the intravenous infusion of secretin and pancreozymin. After the 60 minute control period, calcitonin was infused along with the secretin and pancreozymin. The infusion of calcitonin caused the volume, bicarbonate and enzyme output in the pancreatic juice to decrease to about one-half of that during the control period. During the one hour period, after calcitonin infusion was stopped, the volume, bicarbonate and enzyme content of the pancreatic juice increased but remained slightly lower than that of the control period. The infusion of calcium gluceptate, along with secretin and pancreozymin, overcame the inhibitory effect of calcitonin on pancreatic secretion.

Animals↗
PubMed1977

Early diagnosis of myocardial infarction in the dog with 99mTc-glucoheptonate.

Early gamma imaging of acute experimental myocardial infarcts was evaluated in mongrel dogs with 99mTc-glucoheptonate. From 15 to 20 mCi were injected between 1 and 27 hr after coronary artery occlusion. Nine dogs imaged 3 hr after injection (4 hr after occlusion) showed unequivocal uptake in the region of the infarct. Fifteen dogs imaged 5-7 hr after injection (6-8 hr after occlusion) showed sufficiently well-defined regions of abnormal uptake so that planimetry could be performed reliably. Five animals imaged serially showed improvement of the image only up to about 5-7 hr after injection. Infarct-to-normal myocardium and infarct-to-blood ratios were slightly higher in dogs injected 15-27 hr after infarction than in those injected 1 hr after infarction, implying that equally good results can be obtained with injection and imaging of 99mTc-glucoheptonate at any time within the first day. No other infarct-labeling radiopharmaceutical shares this capability for the early detection a delineation of acute infarcts.

Acute Disease↗